High-density arrays of oligonucleotide probes have been fabricated using spotting technology, spraying technology, electrostatic attraction, and high-resolution photolithography in combination with solid-phase oligonucleotide synthesis. Such forms of DNA detection technology, which are often associated with chip-based structures and microarrays, may be used for parallel DNA hybridisation analysis, directly yielding sequence information from genomic DNA fragments. Prior to sequence identification, the nucleic acid targets are commonly fluorescently labelled. This can occur prior to or after hybridisation to the oligonucleotide array, via direct chemical modification of the target strand or by use of an intercalant or groove-binding dye subsequent to hybridisation on the DNA microarray. The hybridisation pattern, as determined by fluorescence microscopy, is then deconvolved by appropriate chemometric processing to reveal the sequence of the target nucleic acid. Rather than focusing on selective detection of small quantities of a particular nucleic acid sequence as is done in the field of dedicated biosensors, this technology has focused on sequence analysis of nucleic acids in suitably high copy number so as to sufficiently occupy the oligonucleotide array.
Other spatially resolved approaches for development of microarray technologies have also been introduced where electrochemical manipulation of hybridisation at spots or pads of DNA can be done, and where the tips of fibres that form a fibre optic bundle are altered to house addressable discrete DNA microbeads. Further examples of spatially resolved devices include the use of spots of nucleic acids that are deposited onto a glass or fused silica surface by pin spotting or piezo-based ink jets, spatially resolved electrochemical analysis as found in Light-Addressable Potentiometric Analysis (LAPS) technology, and spatially resolved Surface Plasmon Resonance for pads that are located over conductive metals.
In all these cases, the concept is that individual independent spots, beads or pads of nucleic acid are deposited across a surface, and that the immobilized chemistry in each spot, bead or pad is consistent and discrete. In array technologies, each spot, bead or pad typically has a plurality of bound nucleic acid molecules and each spot, bead or pad can contain one or more, although typically a relatively small number of, different bound nucleic acids. The purpose of these arrays is to achieve detection of multiple targets, whether they be pathogenic organisms, mutations or combinations of genes that are concurrently up and down regulated. This is achieved in any one analysis by looking at alterations of a pattern of discrete signals on a surface. The approach is based on study of the results of many partially-selective reactions, where ideally the chemistry of each reaction can be defined and controlled. The problem with such approaches is that it is virtually impossible to select a stringency that is concurrently suitable for optimization of hybridisation at each and every spot, bead or pad, and the approach therefore incorporates a lack of selectivity by design. Furthermore, such detection devices are generally not amenable to providing absolute quantitative results and are not usually reusable.
Approaches to sensor development have basically taken two distinctive paths:
1) The use of one type of ssDNA sequence on a relatively large surface area for biosensor preparation.
2) The use of microarrays of many different ssDNA sequences, each different ssDNA sequence being immobilized in a small, discrete surface area, with many different ssDNA sites being distributed over a large surface area. (More recently, microarrays composed of discrete areas in which a relatively small number of different ss DNA are immobilized have been employed.)
Two common platforms used for development of DNA biosensors are Surface Plasmon Resonance Spectroscopy (SPR) and Total Internal Reflection Fluorescence Spectroscopy (TIRF). SPR can detect surface binding interactions in real time without the use of labels. SPR instrumentation is commercially available and Pharmacia's BIAcore™ instrument is in common use in many laboratories to investigate the kinetics of interfacial nucleic acid hybridisation, formation of triple-stranded complexes, to develop assays for selective detection of polymerase chain reaction (PCR) amplified nucleotides (N. Bianchi, C. Rutigliano, M. Tomassetti, G. Feriotto, F. Zorzato, and R. Gambari, Clinical and Diagnostic Virology 8, pp. 199–208, 1997) and to investigate the use of peptide nucleic acid (PNA) capture probes to enhance selectivity. The BlAcore system has been used by several groups for the monitoring of DNA-DNA interactions in real time (P. Nilsson, B. Persson, M. Uhlin, and P. Nygren, Analytical Biochemistry 224, pp. 400–408, 1995; M. Tosu, M. Gotoh, K. Saito, M. Shimizu, Nucleic Acids Symposium Series 31, pp. 121–122, 1994). The association and dissociation kinetics of target oligonucleotides composed of either complementary sequences or mismatched bases have been monitored. The authors claimed that differences in kinetic parameters could be detected for non-complementary strands as well as for various 20-mers containing two, four or six mismatched base pairs. The time required for each analysis was reported to be 15–20 minutes and the results showed promise for real-time interaction analysis for such processes as gene assembly, DNA polymerase activity, and sequencing experiments. Bier and Scheller (F. F. Bier and F. W. Scheller, Biosensors and Bioelectronics 11, pp. 669–674, 1996) used SPR to study the interaction of the restriction endonuclease EcoRE, a DNA modifying enzyme. The action of the enzyme was observed by measuring the loss of bound DNA after a short incubation with the enzyme.
Numerous evanescent wave fibre optic DNA sensors have been reported in the literature. The evanescent field typically penetrates about 200 nm to 400 nm (typically less than 1 μm) into the surrounding medium when using visible radiation, conferring surface selectivity (W. F. Love, L. J. Button, and R. E. Slovacek, in Biosensors with Fibre Optics. Eds. Wise and Wingard, pp. 139–180, The Humana Press Inc., 1991). The first such fibre optic DNA sensor was reported by Squirrell in 1992 (C. R. Graham, D. Leslie, and D. J. Squirrell, Biosensors and Bioelectronics 7, pp. 487–493, 1992). Preliminary experiments using covalently immobilised probe oligonucleotides and fluorescein-labelled complementary strands gave fast (60 second) detection in the nanomolar range with a linear response curve, but were not as sensitive as radio labelling techniques. Analysis of 204-base oligonucleotides showed that the detection of PCR products was feasible. Abel (A. P. Abel, M. G. Weller, G. L. Duveneck, M. Ehrat, and H. M. Widmer, Analytical Chemistry 68, pp. 2905–2912, 1996) operated a similar system in a competitive binding mode.
Sensitivity of evanescent biosensors may be significantly improved by use of mono-modal optical fibres (T. R. Glass, S. Lackie, and T. Hirschfeld, Applied Optics, 26, pp. 2181–2187, 1987). With use of mono-modal fibres, up to 10% of the optical power may be present in the evanescent field. Bier (F. Kleinjung, F. F. Bier, A. Warsinke, and F. W. Scheller, Anal. Chimica Acta 350, pp. 51–58, 1997), used two strategies for immobilisation of oligonucleotides to monomodal optical fibres: direct coupling to amino-activated surfaces or coupling via the avidin-biotin bridge. Using the fluorescent double-stranded ligands YOYO and picogreen, detection limits of 30 fM (3.2 amol) were achieved. These are the lowest detection limits reported to date for fibre optic DNA biosensors. The sensor was also able to detect single base pair mismatches in the target sequence.
A second major route to production of devices for DNA analysis involves placement of arrays of different sequences across surfaces, or at the tips of fibre-optic bundles (Michael, K. L., Taylor, L. C., Schultz, S. L., Walt, D. R., Anal. Chem. 1998, 70, 1242–1248). Automated oligonucleotide synthesis has seen commercial application by Fodor and Affymetrix (E. L. Sheldon, J. Briggs, R. Bryan, M. Cronin, M. Oval, G. McGall, E. Gentalen, C. G. Miyada, R. Masino, D. Modlin, A. Pease, D. Solas and S. P. A. Fodor, Clinical Chemistry 39, pp. 718–719, 1993; G. H. McGall, A. D. Barone, M. Diggelmann, S. P. A. Fodor, E. Gentalen and N. Ngo, JACS 119, pp. 5081–5090, 1997), where photolithography techniques have been used to grow arrays of oligonucleotides on DNA “chips”. This involves the activation of glass surfaces and then extension of the surface with a hexaethyleneglycol-type linker. The terminal groups of the linker are blocked with photolabile protecting groups. These groups are then removed from predefined regions by selectively exposing the surface with light through photolithographic masks, followed by oligonucleotide addition. This has been done using phosphoramidites with photolabile protecting groups in the 5′-hydroxyl position, or more recently with conventional DMT protected phosphoramidites in combination with polymeric semiconductor photoresist films (G. McGall, J. Labadie, P. Brock, G. Wallraff, T. Nguyen, and W. Hinsberg, PNAS, 93, pp. 13555–13560, 1996). The phosphoramidites react only with the sites that were previously exposed to light. The process is repeated with different lithographic masks until the desired oligonucleotides are obtained. The number of oligonucleotide probes that can be immobilised is limited by the size of the chip and the lithographic resolution (M. Chee, R. Yang, E. Hubbell, A. Berno, X. C. Huang, D. Stem, J. Winkler, D. J. Lockhart, M. S. Morris and S. P. A. Fodor, Science 274, pp. 610–614, 1996). It has been reported that chips with 136,528 unique oligonucleotides have been synthesized on a 13 cm2 chip.
Another approach involves placing aminated polypropylene sheets in a Southern Array Maker (SAM) and then standard phosphoramidite chemistry is applied to 64 distinct and independent channels producing 64 independent oligonucleotides (M. J. O'Donnell-Maloney and D. P. Little, Genetic Analysis: Biomolecular Engineering 13, pp. 151–157, 1996). Other methods involve a piezoelectric ink-jet dispenser that delivers discreet droplets of reagent to chip surfaces, or delivery by “printing” using bundles of capillaries or pins.